Instruction Manual

pH-1 micro
fiber-optic phase detection device

Table of Contents

Instruction Manual
pH-1 micro
Software Version Hview-V5.25b
Oktober 2003
Specification of pH-1 micro:
PC-controlled one-channel fiber-optic
phase detection device for microsensors;
excitation wavelength of 470 nm;
optical fibers of 140 m diameter connected by
ST fiber connectors.

Table of Contents

Table of Contents
1

Preface ..........................................................................................................................1

Safety Guidelines .........................................................................................................2

Description of the pH-1 micro Device .........................................................................4

Extension to a multi-channel multi-analyte system......................................................4

Required Basic Equipment ..........................................................................................7

pH-Sensitive Microsensors: Sensors and Housings .................................................8

5.1

Housings of pH-Sensitive Microsensors ..........................................................11

5.1.1
Needle-type - pH Micro-Sensor (NTH) ..........................................................12
5.1.2
Implantable pH-Micro-Snsor..........................................................................14
Description of pH-1 micro Software..........................................................................17

6.1

Software Installation and Starting the Instrument ...........................................17

6.2

Function and Description of the pH-1 micro Program.....................................18

6.2.1
Menu Bar ......................................................................................................19
6.2.2
Control Bar....................................................................................................24
6.2.3
Graphical Window.........................................................................................30
6.2.4
Status Bar .....................................................................................................31
6.3
Subsequent Data Handling ................................................................................31
7

Calibration and Measurement ...................................................................................33

7.1

Buffers for Calibration........................................................................................33

7.2

Mounting the Implantable Microsensors ..........................................................33

7.3

Mounting the Needle-Type Microsensors .........................................................34

7.4

pHSolver-v07.exe used for calculation of the calibration values....................37

7.5

Calibration/Measurement of a pH Microsensors ..............................................39

7.6

Some Advice for Correct Measurement ............................................................40

7.6.1
Signal drift due to photo-decomposition ........................................................40
7.6.2
Performance proof ........................................................................................41
General Instructions: .................................................................................................42

8.1

Warm-Up Time ....................................................................................................42

8.2

Maintenance........................................................................................................42

8.3

Service.................................................................................................................42

Technical Data ............................................................................................................43

9.1

General Data .......................................................................................................43

9.2

Analog Output and External Trigger .................................................................45

9.3

Technical Notes ..................................................................................................46

9.4

Operation Notes..................................................................................................46

10

Concluding Remarks..................................................................................................47

11

Appendix .....................................................................................................................48

11.1

Basics in Optical Sensing ..................................................................................48

Table of Contents
11.1.1
11.1.2
11.1.3
11.1.4
11.1.5
11.1.6
11.1.7

Major Components of Fiber-Optic Microsensors ...........................................48

Luminescence Decay Time...........................................................................48
Advantages of Optical Lifetime Based Sensors ............................................49
Dynamic Quenching of Luminescence ..........................................................50
Dual Lifetime Referenced (DLR) Optical Sensors .........................................51
Fluorescence (Frster) Energy Transfer (FET) .............................................53
Literature.......................................................................................................54

Preface

Preface

Congratulations!
You have chosen a new innovative technology to read out chemo-optical sensors!
The pH-1 micro is a compact, portable, PC-controlled fiber-optic phase detection device to
read out pH sensors. The data evaluation is PC supported.
The pH-1 micro was specially developed for very small fiber-optic microsensors. It is based
on a novel technology, which creates very stable, internal referenced measured values. The
pH-1 micro is useful to read out sensors with average luminescence decay times in the
range of 1 s to 0.6 ms.
Optical sensors (also called optodes) based on decay time measurements have important
advantages over intensity based optodes:

They are small

They are independent on fluctuations of the light source
Their signal does not depend on the flow rate of the sample
They are less sensitive to photo bleaching and dye leaching
They are not influenced by intensity fluctuations due to fiber bending

Therefore, they are ideally suited for the examination of small sample volumes, long term
measurements in difficult samples, and for biotechnological applications.
A set of different microsensors, flow through cells and integrated sensor systems is available
to make sure you have the sensor which is ideally suited to your application.
Please feel free to contact our service team to find the best solution for your application.
Your Loligo Team.

Safety Guidelines

Safety Guidelines

PLEASE READ THESE INSTRUCTIONS CAREFULLY BEFORE WORKING WITH THIS

INSTRUMENT!
This device has left our works after careful testing of all functions and safety requirements.
The perfect functioning and operational safety of the instrument can only be ensured if the
user observes the usual safety precautions as well as the specific safety guidelines stated in
these operating guidelines.
-

Before connecting the device to the electrical supply network, please ensure that the
operating voltage stated on the power supply corresponds to the mains voltage

The perfect functioning and operational safety of the instrument can only be
maintained under the climatic conditions specified in Chapter 9 "Technical Data" in
this operating manual.

If the instrument is moved from cold to warm surroundings, condense may form and
interfere with the functioning of the instrument. In this event, wait until the
temperature of the instrument reaches room temperature before putting the
instrument back into operation.

Balancing, maintenance and repair work must only be carried out by a suitable
qualified technician, trained by us.

Especially in the case of any damage to current-carrying parts, such as the power
supply cable or the power supply itself, the device must be taken out of operation and
protected against being put back into operation.

If there is any reason to assume that the instrument can no longer be employed
without a risk, it must be set aside and appropriately marked to prevent further use.

The safety of the user may be endangered, e. g., if the instrument

is visibly damaged;
no longer operates as specified;
has been stored under adverse conditions for a lengthy period of time;
has been damaged in transport

If you are in doubt, the instrument should be sent back to the manufacturer for repair
and maintenance.

The operator of this measuring instrument must ensure that the following laws and
guidelines are observed when using dangerous substances:

EEC directives for protective labor legislation;

National protective labor legislation;
Safety regulations for accident prevention;
Safety data-sheets of the chemical manufacturer

The pH-1 micro is not protected against water spray;

The pH-1 micro is not water proof;
The pH-1 micro must not be used under environmental conditions which cause watercondensation in the housing;
The pH-1 micro is sealed;
The pH-1 micro must not be opened;

Safety Guidelines

We explicitly draw your attention to the fact that any damage of the manufactural seal will
render of all guarantee warranties invalid.
Any internal operations on the unit must be carried out by personal explicitly authorized by
Loligo and under antistatic conditions.
Needle-type sensors are housed in extremely sharp syringe needles. Avoid injury by
handling the needle carefully. Please pay attention to all safety guidelines for safe handling
of sharp needles and syringes. Beware of injuring with the needle as well as with the sensor
tip. The glass fiber can break if pricked into the skin and can cause inflammation.
The pH-1 micro may only be operated by qualified personal.
This measuring instrument was developed for use in the laboratory. Thus, we must assume
that, as a result of their professional training and experience, the operators will know the
necessary safety precautions to take when handling chemicals.
Keep the pH-1 micro and the equipment such as PT1000 temperature sensor, power supply
and sensors out of the reach of children!
As the manufacturer of the pH-1 micro, we only consider ourselves responsible for safety
and performance of the device if
the device is strictly used according to the instruction manual and the safety guidelines
the electrical installation of the respective room corresponds to the DIN IEC/VDE
standards.
The pH-1 micro and the sensors must not be used in vivo examinations on humans!
The pH-1 micro and the sensors must not be used for human-diagnostic or therapeutically
purposes!

Description of the pH-1 micro Device

Description of the pH-1 micro Device

The pH-1 micro is a precise single channel pH

meter with fiber-optic pH microsensors based on
2 mm polymer fibers. The small outer
dimensions, low power consumption and a robust
box make it ready for portable and field use. For
operation, a PC/Notebook is required. The pH-1
micro is controlled using a comfortable software,
which also saves and visualizes the measured
values.
The pH-1 micro has 2 analog outputs (0-5 V)
and one trigger input (TTL) to be connected to a
data logger. Analog connectors are BNC
connectors.
The analog outputs are programmable to deliver
pH, temperature, or the raw values (phase shift
or amplitude), and the data are called via
computer and RS232 (digital) or using the
external trigger input (analog).
pH-1 micro instruments features:

high precision
portable (battery power optional)
analog/digital data output
external temperature measurement

Extension to a multi-channel
multi-analyte system
Using a computer port extender providing multiple
RS232 ports, up to eight singles devices (Fibox, Microx
TX, pH-1 micro, ) can be connected to one single
computer. This multi-instrument set-up offers a highly
flexible method to create multi-channel, multi-analyte
measuring systems including additional temperaturecompensation of each channel.

Description of the pH-1 micro Device

Front Panel

ELEMENT

DESCRIPTION

POWER

ON/OFF switch

SENSOR

ST fiber connector

L1

Control
LED

Temp

Connector for PT 1000

temperature sensor

FUNCTION
Switches the device ON and OFF
Connect the fiber-optic sensor here.
red:
instrument off;
green: instrument on;
orange: stand by;
Connect the PT 1000 temperature sensor for
temperature compensated measurements here.

Description of the pH-1 micro Device

Rear Panel of the pH-1 micro device

Two standard BNC connectors are added for analog output channels 1 and 2, another one
for external trigger input.
The electrical specifications of all rear panel connectors are given in technical specification
sheet. Please read also the technical notes to avoid mistakes.

ELEMENT

DESCRPTION

FUNCTION

12 VCD

Line adapter for power

supply

RS 232

RS232 interface
(male)

Connect the device with a RS232 data cable to your

PC/Notebook here.

CH 1

Analog out
(channel 1)

Connect the device with external devices, e.g. a data

logger

CH 2

Analog out
(channel 2)

Connect the device with external devices, e.g. a data

logger

EXT TRIG

External trigger input

Connector for 9 - 36 V DC power supply.

Connect the device with external devices, e.g. data

logger with a trigger output, pulse generator

Required Basic Equipment

Required Basic Equipment

pH meter pH-1 micro*

Software Hview for pH-1 micro*
PC / Notebook
(System requirements:
Windows 95/98/2000/Millenium/NT 4.0/XP; Pentium processor, at least 133 MHz, 16 MB
RAM)
RS 232 Cable *
Line adapter (110 - 220 V AC, 12 V DC) *
Temperature sensor PT 1000*
Analyte-sensitive microsensor
The sensors are mounted into different types of housings

Vessels for calibration solutions

We recommend Schott laboratory bottles with a thread which can be obtained by VWR International
(ordering number: 215L1515)

Laboratory support with clamp, sensor-manipulator

*: scope of supply

Fiber-Optic Microsensors: Sensors and Housings

pH-Sensitive Microsensors: Sensors and Housings

Optical pH Sensors designed for pH-1 micro device are based on the new Dual Luminophor
Reference method (see also appendix). They consist of an inert long decay time reference
dye and a short decay time indicator dye, which changes its fluorescence intensity due to the
pH-value. The measured value, the average decay time, represents the ratio of the two
fluorescence intensities. Therefore, the signal is internal referenced and insensitive to
fluctuations of the light source.
Dynamic range
Optical pH sensors respond according to the mass acting law, hence, they do not show a
linear behavior like potentiometric pH electrodes. This limits their use to a dynamic range of
approximately 3 to 4 pH units. On the other hand, the resolution can be very high in the
optimal range. Figure 5.1 shows a typical response curve of a pH sensor. Increasing the pH,
the signal - in our case the phase angle - decreases. The phase angle can be related to
the pH as shown in Figure 5.2. The theoretical aspects are explained more detailed in the
appendix.

10

60

55

50
45
phase

7
pH

Imax
Imin
dpH
pH0

6
5

40

: 18.77
: 56.33
: 00.64
: 06.51

35
30

25

20
0

time, min

15
4

pH

Figure 5.1 Response of pH micro-sensor pH-HP-5.

Figure 5.2 Effect of the phase angle of a pH

microsensor (pH-HP-5) on different pH values.

The characteristic of a typical sensor are listed below:

Dynamic range: pH 5.5 to 8.5
Resolution: 0.01 pH
Drift due to bleaching: 0.03 per 1000 measuring points
Response time: less then 30 sec
Please note that the given values may differ from your special device and sensoric
application.

Fiber-Optic Microsensors: Sensors and Housings

Resolution

7.00
Continous mode (measurement each second)

6.95
pH

6.90

6.85

6.80
0.00

2.00

4.00

6.00

8.00

10.00

time, min

Figure 5.3. Resolution of the pH sensor can be up to 0.01 pH

Cross sensitivities
While pH electrodes are influenced by sulfide, electromagnetic fields or flow velocity the
optical pH measurement is interfered by ionic strength. This problem can be overcome by a
calibration with buffers of similar ionic strength than the sample.

55
50
45

is50
is100
is200

phase

40
35
30
25
20
15
4

pH

Figure 5.4 Cross-sensitivity to ionic strength we recommend to calibrate the sensor in pH-adjusted
media!

10

Fiber-Optic Microsensors: Sensors and Housings

Sensor drift

Like all optical chemo sensors, optical pH-sensors are suspect to photobleaching. Due to
this process the measured intensity (amplitude) is decreasing while the phase of the sensor
is increasing. This process is quite slow and depends on the used light intensity and the
duration of illumination. If you are in doubt whether a measured increase in phase is due to
photobleaching or decreasing pH of the sample, please lower the light intensity. If the drift is
not getting smaller, your signal drift is due to pH changes in your sample.

8
7.8
7.6
7.4
7.2
pH 7
6.8
6.6
6.4
6.2
6
0

200

400

600
800
Measuring points

1000

1200

1400

Figure 5.5 Drift due to bleaching: 0.03 per 1000 measuring points
Typical bleaching rates are listed below.

1000 Measuring points

1 hour in sec. Mode
1 d in 1 min Mode

Sensor Drift due to Bleaching

0.03 pH
0.1 pH
0.05 pH

Please notice, that bleaching rates also depend on the adjusted light intensity of the LED and the
individual instrument and sensor.

Temperature dependency
pH Micro sensors display, as any other pH sensors, a distinct temperature sensitivity. Some
typical calibration curves at different temperatures are shown below:

11

Fiber-Optic Microsensors: Sensors and Housings

55
50

5 C
15 C
20 C
30 C
35 C
40 C

45

Phase

40
35
30
25
20
15
4

10

pH

To obtain most reliable results we recommend to calibrate at the same temperature as used
for measurement. Higher temperature mimics lower pH. At pH 7 a deviation of about 0.08 ph
/ 5 C occurs. Please contact our service team for temperature compensation formulas for
calibration constants.
Limitations
The offered pH sensors for pH-1 micro were specially designed for physiological samples
and media. Samples with extremely low ionic strength and low buffer content may be not
measured correct. To test whether the sample can be measured correct, try to perform the
calibration procedure with a buffer system which is as similar to the sample as possible. If
the calibration procedure does not result in a sigmoidal shaped calibration plot, the system is
not suited for the sensor. Please contact our service team for special sensors.
The measurement can also be influenced by small, highly fluorescent molecules like
fluorescein or rhodamin in the sample.
The sensors do not stand pH above 9 for prolonged time and organic solvents.

Response time
The response time (t90) of the pH sensor is dependent from the diffusion rate of protons
through the sensor layer, hence, the response time is dependent from the thickness of the
sensor layer and the stirring rate. The typical response (t90) time of a pH-micro sensor is
below 30 sec.

5.1

Housings of pH-Sensitive Microsensors

The fiber-optic pH microsensors are based on 140 m silica optical fibers. To protect the
small glass fiber tip against breaking, suitable housings and tubings around it, depending on
the respective application, were designed.

Fiber-Optic Microsensors: Sensors and Housings

12

Of course, it is possible to build customer-specific housings. Please feel free to contact our
service team to find the best solution for your application.
We offer the following standard designs:

5.1.1

Needle-type - pH Micro-Sensor (NTH)

Needle-type pH micro-sensors are miniaturized

fiber optic chemical sensors designed for all
research applications were a small tip size of
ca. 140 m are necessary. Needle-type pH
micro-sensors are ideal for pH profiling in
sediment and biofilms with a high spatial
resolution. The pH-sensitive tip of the optical
fiber is protected inside a stainless steel needle.
Such a design is the best for an easy
penetration through a septum rubber or any
other harsh material as well as secure for
transportation. For measurement purposes the
sensor must be pushed out from the needle.

Schematic drawing of the NTH (Needle-type) micro-sensor

Fiber-Optic Microsensors: Sensors and Housings

male fiber-optic plug

spring
safety nut

fiber cable

syringe plunger

~
needle plastic base

syringe housing
syringe needle
transport block

needle plastic base

glass fiber

protective plastic cap

sensor tip
syringe needle

Features

High spatial resolution (ca. 140 m)

Penetration probe for insertion into semisolids like sediments or biofilms
Penetration through septa
Easy to handle
Different needle size available
No reference electrode needed
High temporal resolution
Insensible towards electrical interferences and magnetic fields

Suitable for pH-1 micro and pH-4 micro

Specification
Tip size

140 m

Measuring range

5 9 pH

Response time

30 sec

Resolution

Up to 0.01 pH

Drift

0.03 pH

13

Fiber-Optic Microsensors: Sensors and Housings

14

(per 1000 Measuring points)

Temperature Range

from 5 C to + 50 C

Cross-Sensitivity

Optical pH sensors display slight cross sensitivity to

ionic strength (salinity) and towards small
fluorescent molecules like dissolved indicator dyes.

ORDER
The NTH (Needle-type) pH sensors are offered with options to be specified in the purchase order
form. The order code key shown below (see also the example) defines the parameters. Please,
choose the parameters that best meet Your requirements.

NTH

pH

HP5

NS

Code of pHNeedle-Type
sensor coating
Housing Analyte pH
Length of
Glass Fiber
- 2.5 m
-5m
- 10 m

Stainless Needle
Length [mm] / diameter [mm]
- 20 / 0.4
- 40 / 0.4
- 40 / 1.2
- 120 / 0.8

Example

NTH

pH

HP5

NS 40 0.4

With this code you will order a microsensor mounted in a needle-type housing (NTH), with the pH
sensitive coating HP5 with a glass fiber length of 5 m (L5) mounted in a stainless needle of 40 mm
length and 0.4 mm diameter (NS 40/0.4) .

5.1.2

Implantable pH-Micro-Sensor

Implantable pH micro-sensors are the

miniaturized fiber optic chemical sensor
designed for all research applications were a
small tip size of 140 m are necessary.
Implantable pH micro-sensors are ideal for
costumer-specific set up. They were applied
for implantation in blood circuits and for
profiling with a high spatial resolution

Fiber-Optic Microsensors: Sensors and Housings

15

Schematic drawing of the Implantable micro-sensor

FEATURES

High spatial resolution (ca. 140 m)

Easy to handle

Most flexible for a wide range of applications

High temporal resolution

Insensible towards electrical interferences and magnetic fields

This implantable micro-sensor can be used in combination with the pH meter pH-1 micro
and pH-4 micro
SPECIFICATION
Tip size

140 m

Measuring range

5 9 pH

Response time

30 sec

Resolution

Up to 0.01 pH

Drift
(per 1000
points)

Measuring 0.03 pH

Temperature Range

from 5 C to + 50 C

Cross-Sensitivity

Optical pH sensors display

slight cross sensitivity to ionic
strength
(salinity)
and
towards small fluorescent
molecules
like
dissolved
indicator dyes.

16

Fiber-Optic Microsensors: Sensors and Housings

ORDER
The Implantable pH sensors are offered with options to be specified in the purchase order
form. The order code key shown below (see also the example) defines the parameters.
Please, choose the parameters that best meet Your requirements.

IMP
Implantable
Housing

pH

HP5

900/

600/

140/

inner plastic cable

Code of pHdiameter [m] / length [cm]
sensor coating
600 m / 0 to customer request
Analyte pH
outer plastic cable
length of bare glass fiber
Diameter [m] / length [m]
Diameter [m] / length [mm]
900 m / 0 10
140 m / 1 to customer request

Example

IMP

pH

HP5

900/ 5

600/ 1

140/ 2

This is the order code for the implantable micro-sensor (IMP) with the pH sensitive coating
HP5, with a fiber cable length of 5 m (900/5), a cable plastic jacket length of 1 cm (600/1), a
bare optical fiber length of 2 mm (140/2)

Description of pH-1 micro Software

17

Description of pH-1 micro Software

This software is compatible with Windows 95/98/2000/Millenium/NT4.0/XP.

6.1
1.

2.
3.
4.

5.

Software Installation and Starting the Instrument

Insert the supplied disc/CD into the respective drive. Copy the file Hview525b.exe onto
your hard disk. (for example, create C:\PDD\Hview525b.exe). Additionally, you may
create a link (Icon) on your desktop.
Connect the pH-1 micro via the supplied serial cable to a serial port of your computer.
Tighten the cable with the screws on your computer and on the pH-1 micro.
Connect the power supply.
Please close all other applications as they may interfere with the software. Start the
program Hview525b.exe with a double click. The following information window appears:

If the right com port is adjusted this information window disappears within a few
seconds. If the wrong com port is adjusted you are asked to set the right com port:

With a right mouse click onto com port you are able to set the right com port. Please
confirm your selection by clicking the OK button. The information window disappears if
the right com port is adjusted.

Description of pH-1 micro Software

6.2

Function and Description of the pH-1 micro Program

The window shown below is displayed after starting the software Hview525b.exe:
The program has 4 main sections:
1. Menu bar
2. Graphical window
3. Status bar
4. Control bar, divided into numerical display, control buttons and warning lights
menu bar
control buttons
warning lights

numerical display

graphical window

Description of pH-1 micro Software

6.2.1

Menu Bar
File

Exit

Charts
pH

Display
Zoom
AutoScaleY1
Undo Zoom

Temperature
Amplitude
Phase
Print
Charts

Clear Charts
Dimensions

Settings
Com Port
Instrument Info
analog settings
LED Intensity
Frequency

File
Exit
Closes the program.
Charts
The respective charts of the measurement can be displayed (v) or hidden
pH:
The measured pH value
Temperature:
The measured temperature
Amplitude:
The magnitude of the sensor signal
Phase:
Phase angle, the raw data
Display
Zoom:

AutoScaleY1 is the default setting. AutoScaleY1 means that the y-axis is scaled
automatically.
Undo Zoom: The original display is recovered; see also graphical display

Clear Charts: The graphs shown on the display is cleared.

19

Description of pH-1 micro Software

20

Dimensions:
You can adjust the number of
measurements points on the x-axis
shown in the display (maximum
number of points are 5000)
Furthermore, you can adjust the
micromum and the maximum of the yaxis.
The AutoScaleY1 function is switched
off.

Print
Charts: The charts shown in the display can be printed
Settings
ComPort
The serial comport (com1 com20) for the serial interface (RS 232) can be chosen in this
window. COM 1 is the default setting. If you choose the wrong Com port, the information
window Connect the instrument to the PC and choose the right com-port does not
disappear.
Instrument Info:
Here you can find the version of the software and some important settings of the instrument.
If you have a problem with the pH-1 micro device, please contact our service team and have
the software and instrument information ready.
To change back to the graphical window click the Measure Chart button.

Description of pH-1 micro Software

Instrument Info

Software Info

21

Description of pH-1 micro Software

22

LED-Intensity
With the current of the LED you can adjust the amount of light illuminating the sensor.
You can choose between an Auto Adjust of the LED where the pH-1 micro adjusts the
optimal LED current itself, or you can select Advanced where you can adjust the LED
current yourself.
If you increase the LED current, the signal amplitude increases, since a higher light density
illuminates the sensor. Higher amplitudes lead to more stable values and therefore higher
resolution, but also to increased photobleaching.
Auto Adjust:
To make the adjustment of the LED intensity automatically, just click the button Start Auto
Adjust. Please check that the Microsensors has been connected to the instrument.

The automatically adjustment of the LED intensity is finished when in the status window the
message Auto adjustment finished appears. Click the Close button to confirm the
settings.

Description of pH-1 micro Software

23

Advanced:
Click the Advanced button to change the LED current manually. Values between 10 and
100 % are possible. After clicking the confirm button you can see the change of the
amplitude in the window below.

Please note:
By increasing the light intensity you increase the amplitude of the sensor. This leads to
smoother phase signals. However, increasing the light intensity can increase
photobleaching, which decreases the shelf-life of your sensor.
Frequency - This option is only for advanced users with special adapted sensors.
Please refer to the special advice of our service team.

With the menu frequency you can adjust the optimal modulation frequency of your analytesensitive indicator dye. Please chose 49: 44.86 kHz for the pH sensor pH-HP5.
If you change the settings you have to restart the software.

Description of pH-1 micro Software

24

Analog output
Here you can choose which data are exported via the analog output. The pH-1 micro device
has two analog outputs and one trigger input. The desired data sources (temperature,
amplitude, phase) can be chosen via the dialog box.
Equivalence coefficient
temperature
amplitude
phase

6.2.2

1 : 0.1 (e.g. 208mV = 20.8C)

1 : 20 (e.g. 1110mV = 22200 relative units)
1 : 0.025 (e.g. 1100mV = 27.50)

Control Bar

Numerical display

The measured pH is shown in the display window.

Description of pH-1 micro Software

25

Temperature measurement:
The actual temperature value of the sample (in the case of temperature compensated
measurements) is displayed in the temperature window.
If measurement is performed without temperature compensation, the manual inserted
temperature is displayed with the hint that temperature measurement is offline.
Control buttons:
The way to start a measurement is
(1) Calibration of the sensor by input of calibration values
(2) Start Measurement with Assistant
(3) Log Data

Calibration:

Type in the calibration values. Calibration values are obtained using DLRpH.xls. Please see
7.1 Calibration/Measurement of a pH Microsensors using the File DLRpH.xls for Details

Description of pH-1 micro Software

26

Measurement:
The measurement assistant is opened (default setting).
Quick Start:
The measurement is started. The measurement settings are continuous mode which means
that each second a new measurement data is recorded. The measurement is temperature
compensated i.e. a temperature sensor has to be connected. If no temperature sensor is
connected the following warning window appears.

Click the Close button if you want to continue the measurement without temperature
compensation. The temperature is set to 20 C by the software. Connect the temperature
sensor if you want to perform a temperature compensated measurement.
If you want to change the measurement settings click the Advanced start button.
Please note:
The measurement values are not stored. Click the Log Data button to store the
measurement data.
Advanced Start:
In the Advanced Start mode it is possible to adjust user-defined measurement settings.

In the Sampling Rate window you can select the desired measurement mode with a dropdown menu.

Description of pH-1 micro Software

27

By clicking the drop down menu you can choose from fast sampling (update rate each 250
450 ms) to the 60 min mode where each hour a measuring point is recorded.
The speed of recording a measurement point in the fast sampling mode is about 250 ms
when no temperature sensor is connected and the analog output settings are switched off
and decreases to about 450 ms when connecting a temperature sensor or activating the
analog output channels.

Please note:
The sensor shelf life can be increased using a slower measuring mode since the effect of
photo-bleaching is reduced. The illumination light is switched off between sampling. A further
advantage using a high measuring mode is that huge amounts of data for long-time
measurement can be avoided.

Dynamic averaging
The dynamic average defines number of averaged
measured values. The higher the running average, the
longer the time (sampling time) used for averaging. The
higher the running average is set, the smoother the
measurement signal (maximum 25 samples); The
default setting is 4.
Additional temperature measurements
In the temperature compensation window you can decide whether you want to measure
with or without temperature measurement.
If you want to measure with the additional temperature sensor Pt1000, click the on button.
Please ensure that the temperature sensor Pt1000 is connected to the pH-1 micro, before
you click the Start button to continue. The window where you can enter the temperature
manually is disabled.

Description of pH-1 micro Software

28

If you want to measure without temperature compensation, choose the off button. You will
now be requested to enter the temperature of the sample manually. Click the Start button
to start the measurement.

Log Data:
To store the data of your measurement click the Log Data item. Next to the Log Data item
an information window displays whether the actual measurement is stored to a file (logging)
or not (no logging);

The measurement description which you can enter in the text field Enter a description to
the header of the file is stored in the Ascii File.
By clicking the button Choose File, you can select the location where you want to store the
data. Choose as file extension *.txt. Click the speichern button to confirm your settings.

Description of pH-1 micro Software

29

By clicking the Stop Log Data item you stop data logging which is displayed by the blinking
no logging in the information window next to it.

Stop Measurement
The measurement is ended by a left click on the stop button in the control bar.

Description of pH-1 micro Software

30

Warning Lights:
At the right bottom of the window you can find the amplitude, phase angle and three warning
lights. The warning lights are explained below:

amplitude:

red:

green:

Amplitude is too low, the sensor tip may be damaged or sensor

cable may not be connected
Amplitude is critically low, replacement of the sensor is
recommended
amplitude is correct

red:
green:

phase angle is out of limits

phase angle is in normal range

yellow:

phase:

ambient light: red:

green:

background light (e.g. direct sunlight, lamp) is too high. Decrease

of false light is recommended
ratio of sensor signal to false light is acceptable

By clicking the Display Raw Values button, the raw data of

phase angle and amplitude are displayed next to the warning
lights.

6.2.3

Graphical Window

The respective sensor signal is displayed according to the selection of the 4 control buttons
pH, phase, amplitude and temperature (menu chart). The raw values (the phase angle in
degrees and the sensor amplitude in mV) can also be displayed by clicking the button
Display Raw values. The temperature is given in [C]
Zoom Function:
1. Press the left mouse button and drag from left to right to enlarge a certain area of the
graphical window. The graphical window displays the selected data points and is not
actualized with new data.
2. Press the left mouse button and drag from right to left to recover the original display, or
click the Undo Zoom button in the display menu under zoom.

31

Description of pH-1 micro Software

6.2.4

Status Bar

sw

sw

sw

sw

sw1: Displays the serial port which is used for communication of the pH-1 micro device
with the PC
sw2: Displays the file name in which the measurement data are stored. No storage file
selected is displayed if no file was selected (no data storage).
sw3: Displays the start time of the measurement
sw4: Displays the actual time

6.3

Subsequent Data Handling

In the head of the ASCII file, you find the description of your measurement which you have
entered by storing the file.
Below you find the instrument info containing all important settings of the instrument and
firmware and the calibration values and the date of the calibration
.
The software info below contains the version number of the pH-1 micro software, date and
time of the performed measurement. If there is a problem with the pH-1 micro device, please
contact our service team and have the software and instrument information ready.
Below, you find the measure mode settings containing the dynamic averaging, and the
measuring mode.
The following rows, separated by semicolons, list the measuring data. The first two rows
contain the date and time, the third the log-time in minutes. The pH values are stored in the
fourth row. The raw data - phase angle in [] and the amplitude in [mV] - are stored in the
fifth and sixth row, respectively. The seventh row contains the temperature in C measured
by PT1000 temperature sensor. Raw data can be used for user defined recalculations
according to the formulas and tables of the delivered Excel sheet
The eigthth raw of the Ascii file displays possible error messages.

32

Description of pH-1 micro Software

***** DESCRIPTION ***********

pdd3470 1004 03 007 mit gestecktem sensor
***** INSTRUMENT INFO *******
IDENTIFICATION
PHIboard number : 20030007
PM number
: 20030110
Serial number : -PDD3470-1004-03-007
MUX channel
: OFF - 00
PARAMETERS
Signal LED current: 050
Ref LED current : 058
Ref LED amplitude : 88397
Frequency
: 049
Sending interval : 0001
Averaging
:5
Internal temp : 26.8 C
SYSTEM SETTINGS
APL function
: OFF
Temp compensation : ON - ch a
Analog out
: chA t chB t
RS232 echo
: ON
CALIBRATION
Sensor type
:1
Imax
: 22.00
Imin
: 54.00
dpH
: 00.66
pH0
: 07.00
Date (ddmmyy) : 241003
Pressure (mBar) : 0020
FIRMWARE
Code v1.088PH IAP: 07/31/03, 10:45:01
Xilinx built
: 20/08/02 (MM/DD/YY)
Reset condition : SLEEP

***** SOFTWARE INFO *********

HView - V5.25b 09/2003
24.10.2003
10:43:16
******MEASURE MODE SETTINGS**
Dynamic Averaging
4
measure mode
1 sec
start time

10:37:44

date(DD/MM/YY)
time/hh:mm:ss logtime/min pH
24.10.2003 10:43:16
0
24.10.2003 10:43:17
0.017
24.10.2003 10:43:19
0.034
24.10.2003 10:43:20
0.051
24.10.2003 10:43:21
0.068
24.10.2003 10:43:22
0.086
24.10.2003 10:43:23
0.103
24.10.2003 10:43:24
0.12
24.10.2003 10:43:25
0.137
24.10.2003 10:43:26
0.154
24.10.2003 10:43:27
0.171
24.10.2003 10:43:28
0.188
24.10.2003 10:43:29
0.205

phase/
5.09
5.075
5.068
5.08
5.08
5.07
5.105
5.105
5.085
5.098
5.093
5.128
5.12

amp
52.3
52.34
52.35
52.32
52.32
52.35
52.27
52.26
52.31
52.28
52.29
52.2
52.22

temp/C
3464
3475
3487
3461
3477
3494
3479
3468
3454
3464
3468
3481
3465

ErrorMessage
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0

Measurement

33

Calibration and Measurement

Calibration of the sensors is recommended before each measurement. Each calibration is

only valid for the corresponding sensor and should be repeated before beginning a new
measurement. Especially, after longer measurements (more than 1000 measuring points)
the sensor should be re-calibrated.

7.1

Buffers for Calibration

For the calibration of pH Micro-sensors at least 4 different buffers are needed. To obtain
best results a similar composition as the measured sample is recommended. For example,
for measurement in cell culture media calibration in buffers with an ionic strength of 140 mM
and a phenol red concentration of 15 mg/l is ideal. Please notice, that the pH range of the
calibration should exceed the pH range of the measurement e.g. it is not favorable to
calibrate with buffers of pH 4.0, 5.0, 5.5, and 6.0 and to measure about pH 7. Calibration is
only correct for interpolation, not for extrapolation. Please contact our service team for
detailed description of the preparation of calibration buffers or for customized calibration.

7.2

Mounting the Implantable Microsensors

1.

Remove the microsensor carefully from the protective cover. The microsensor is
protected with a glass housing during the transport.

2.

Fix the glass housing microsensor with a clip to a laboratory support or a similar stable
construction.

Measurement

34

We strongly advise you not to handle with microsensors without the support especially when the sensor tip is extended.
3.

Remove the protective cap from the male fiber plug and connect it to the ST-plug of the
Microx TX3 device. The female fiber-plug of the Microx TX3 has a groove in which the
spring of the male fiber-plug of the microsensor has to be inserted. The safety nut must
be carefully attached while turning and is locked by turning slightly clockwise. Be careful
not to snap off the fiber cable.

ST-connector

male fiber plug

7.3

Mounting the Needle-Type Microsensors

1.

Remove the microsensor carefully from the protective cover. The needle-type
microsensor is housed in 0.4 x 40 mm syringe needle mounted to a 1 mL plastic syringe
housing with integrated PUSH & PULL - IN & OUT mechanism. The syringe needle is
protected with a protective plastic cap (A).

2.

Carefully remove the protective plastic cap (A) covering the syringe needle.
When doing so, grip the plastic base of the needle tightly. The syringe needle must not
be removed from the syringe housing. Work carefully!

35

Measurement

tightly grip
the needle base

3.

Fix the microsensor with a clip to a laboratory support or a similar stable construction.

We expressly warn you not to handle with microsensors without the support especially when the sensor tip is extended.
4.

Remove the protective cap from the male fiber plug and connect it to the ST-plug of the
pH-1 micro device. The female fiber-plug of the pH-1 micro has a groove in which the
spring of the male fiber-plug of the microsensor has to be inserted. The safety nut must

Measurement

36

be carefully attached while turning and is locked by turning slightly clockwise. Be careful
not to snap off the fiber cable.

ST-connector

male fiber plug

5.

The glass fiber with its sensing tip is prevented from slipping using a transport block (B).
Remove the transport block from the hole in the syringe housing. Now it is possible to
retract or extend the glass fiber with its sensor tip by pushing or pulling the plunger.
Before pushing out the sensor tip, make sure that you have removed the protective
plastic cap and have some space in front of the syringe needle.

sensor tip

Measurement

37

WHEN GLASS-FIBER WITH ITS SENSOR TIP IS PUSHED OUT, HANDLE WITH CARE.
THE GLASS FIBER IS UNPROTECTED AND MIGHT BREAK

7.4

pHSolver-v07.exe used for calculation of the calibration values

To receive pH values it is necessary to determine calibration values of the sensor. This can
be done by the use of pHSolver-v07.exe. Other fitting programs (e.g. Origin or MathLab)
which can handle the Boltzman equation can be used too.
The delivered File pHSolver-v07.exe does all the calculations. It calculates a Boltzman
curve fit for the measured values.
Ensure that dot instead of comma is used as decimal separation (US-Standard)
1)

Copy the files libraryfiles.exe and pHSolver-v07.exe to a folder named pH-solver.

2)

Start libraryfiles.exe by doubleclick and follow the instructions. If the software asks you
if an existing file should be overwritten, please click on NO.

3)

Start the software pHsolver-v07 by double click.

4)

Type in the calibration values of the sensor data sheet into the respective "inital value"
fields.

Measurement

5)

38

Type in the pH-phase value pairs of your calibration (first pH value of the buffer, then
space, then phase, enter, next pair.) Use at least 5 values.

Type in calibration values

Or use the paste data function (for example copy a set of data of a excel sheet).

Or open a data file containing the data in the format:

Measurement

6)

7.5

39

Click on "least square fit" and you will get the set of calibration data in the calibration
result area. You can store the data in a file by pressing transfer data. If the "initial
guess" is far from the real data, the software may ask you to try again - please just
click on yes, the software will find a new guess on its own.

Calibration/Measurement of a pH Microsensors

1.

Connect the pH-1 micro via the RS232 cable to your computer.

2.

Switch on the pH-1 micro and connect the sensor as shown in Chapters 7.2 to 7.3
depending on the sensor in use.

3.

Start the pH-1 micro software on your computer and start the measurement (see
chapter 6.2.2).

4.

Calibrate the pH sensor, pH-sensitive foil with the calibration solutions (e.g. buffer
solutions of pH 5, pH 6, pH 7, and pH 8). To minimize the response time, slightly stir the
buffer solution. Please ensure that the sensor is dipped in the buffer solution.

5.

Wait about 3 minutes until the phase angle belonging the respective buffer is constant
(the variation of the phase angle should be smaller than 0.1)

6.

Wash the sensor tip with distilled water to clean it from respective buffer solution before
taking the next calibration value.

7.

Open the file pHSolver-v07.exe and enter the pH-values of the used buffer (i.e. 4.0, 5.0,
6.0, 7.0, 8.0 and 9.0) and the corresponding phase values in the calibration value
window (see above).

8.

Press the "least square fit button.

9.

The calibration results phasemax, phasemin, dpH, pH0 are shown and the calibration curve
is displayed.

Measurement

40

10. Wash the sensor with distilled water to clean it from buffer components.

7.6
7.6.1

Some Advice for Correct Measurement

Signal drift due to photo-decomposition

pH sensor-sensors are suspect to photobleaching which results in a drift of the sensor to

lower pH. Photobleaching is higher at basic pH. Photobleaching depends at the amount of
light. Therefore you should reduce the amount of light. The pH-1 micro device offers two
ways of reducing the amount of light.
First, you can lower the amount of measuring points in the case of long term measurements
by choosing the second or minute mode in the measuring assistant (see chapter 6.2.2.
Control Bar, Advanced Measurement)
Second, you can lower the LED current:
Choose LED-Intensity in the Settings menu of the menu bar.
With the current of the LED you can adjust the amount of light illuminating the sensor spot.
By increasing the light intensity you increase the amplitude of the sensor. This leads to
smoother phase signals. However, increasing the light intensity can increase
photobleaching, which decreases the shelf-life of your sensor.
Bleaching always mimic lower pH. For long term measurements the drift can be estimated by
the amount of measurement points and LED intensity. The sensor should be recalibrated
after the measurement and the calibration values compared to the initial values to obtain
maximum accuracy.
Please notice, that the noise (and therefore the resolution) of the signal depends on the
amplitude of the sensor. Lowering the LED current will lead to lower photobleaching but
increased noise of the sensor. The best resolution is achieved if the amplitude of the sensor
is more then 5000. An amplitude of less then 2000 will result in a high noise of up to 0.1 pH.

41

Measurement

7.00
Continous mode (measurement each second)

6.95

pH

6.90

phase/ Led100
phase/ LED 15

6.85

Linear ( phase/ Led100)

6.80
0.00

Linear ( phase/ LED 15)

2.00

4.00

6.00

8.00

10.00

time, min
Figure 7.3 Phase resolution illumination the sensor tip with 15% and with 100 % LED current,
respectively.

7.6.2

Performance proof

If you want to prove the performance during the past measurement, please check the
calibration buffers by inserting the sensor tip in the buffer solution pH 6 and pH 7 when you
have finished your measurement. If the device displays the correct pH values, the sensor
worked perfectly during the whole measurement.
If you are in doubt whether the sensor has to be changed, please follow this procedure:
1) Expose the sensor to pH 4 Buffer solution (e.g. Certipur Buffersolution pH 4, Merck,
109435) and adjust intensity to a value of more then 2000 (ideally more then 10000)
by adjusting the LED current (see page 20). If this is not possible and the fiber cable
and connection to the sensor is OK, change the sensor. The measured phase must
be between 60 and 48 - otherwise check frequency (must be 049) or change
sensor.
2) Expose the sensor to pH 9 Buffer solution (e.g. Certipur Buffersolution pH 9,
Merck, 109461). The measured phase must be between 35 and 10 - otherwise
change sensor.

General Instructions

General Instructions:

8.1

Warm-Up Time

42

The warm up time of the electronic and opto-electronic components of the pH-1 micro is
5 min. Afterwards stable measuring values are obtained.

8.2

Maintenance

The instrument is maintenance-free.

The housing should be cleaned only with a moist cloth. Avoid any moisture entering the
housing! Never use benzine, acetone, alcohol or other organic solvents.
The ST-fiber connector of the sensor can be cleaned only with lint-free cloth. The sensor tip
may be rinsed only with distilled water. Please ensure, that no sample residues are inside
the syringe needle. If necessary, rinse the glass-fiber with distilled water.

8.3

Service

Balancing, maintenance and repair work may only be carried out by the manufacturer:
Loligo Systems ApS
Niels Pedersens All 2
DK-8830 Tjele
Denmark
Phone: +45 8999 2545
E-mail: mail@loligosystems.com
Internet: www.loligosystems.com

Please contact our service team should you have any questions. We look forward for helping
you and are open for any questions and criticism.

43

Technical Data

Technical Data

9.1

General Data

MODES
pH

range: 4.5 8.5

resolution: 0.01 pH units

Electrical temperature sensor

range: 0 - 50 C

(Pt 1000)

resolution: 0.5 C
accuracy: 2 C

OPTICAL OUTPUT / INPUT

Optical connector

ST compatible, Core/Center 100/140

Channels

Wavelength

470 nm

TEMPERATURE SENSOR INPUT

Lemo Connector Size 00

DC INPUT

Connector for Pt-1000 temperature sensor

DC-Range :
12 V/1250mA up to 18V/900mA

Technical Data

44

DIGITAL OUTPUT
communication protocoll

serial interface RS232

19200 Baud, Databits 8, Stoppbits 1, Parity none, Handshake
none

instrument output:

on RJ11 4/4 plug

Interface cable to PC:

RJ11 4/4 to DSub9:

ENVIRONMENTAL CONDITIONS
Operating temperature
Storage temperature

0 to +50C
-10 to +65C

Relative humidity:

up to 95%

OPERATION CONTROL

LED at the front panel:

red:
instrument off
green: instrument on
orange: stand by

DIMENSIONS

length: 185 mm;

width: 110 mm;
height: 45 mm;
weight: 630 g;

Technical Data

9.2

45

Analog Output and External Trigger

The TX3-AOT instrument version is supplied with a dual programmable 12bit analog output
with galvanic isolation and an external trigger input.

ANALOG OUTPUT

GENERAL SPECIFICATION - ANALOG OUTPUT

Channels
Connector
Resolution
Output range
Galvanic isolation
Shortcut protection

2
BNC
12 bit
0 to 4095mV (2mV max. error)
500V rms
Yes

Programmable to

temperature, amplitude, phase by software

Equivalence coefficients :
temperature
1 :: 0.1
amplitude
1 :: 10
phase
1 :: 0.025

(i.e. : 208 mV = 20.8C)

(i.e. : 2220 mV = 22200 relative units)
(i.e. : 1100 mV = 27.50)

Update rate:
The update rate is dependent on the sampling rate of the software.
If an external trigger is used, the update rate is equivalent to the trigger pulse rate.
DC SPECIFICATION - ANALOG OUTPUT
Resolution
temperature
amplitude
phase

2mV 0.2C
2mV 20 relative units
2mV 0.05)

Accuracy error

10mV

Technical Data

46

EXTERNAL TRIGGER INPUT

GENERAL SPECIFICATION Channels

Connector
Input voltage range
Trigger mode
Normal state
Isolation

EXTERNAL TRIGGER INPUT

1
BNC
TTL-compatible / up to 24V
Low-High-Low
(Input must be kept Low for at least 50s)
no current
500V rms

Timing Specifications:
Min rise &fall time for trigger
Max rise &fall time for trigger
Min pulse length
Min pause length
Min periode length

9.3

15ns (see TTL-specification)

2 ms
3 ms
10 ms
13 ms

Technical Notes

Power Adapter
pH-1 micro should always be used with the original power adapter (110-220VAC/12VDC). As
an alternative power source a battery can be used that meets the DC input voltage given in
technical specification. The battery adapter cable is available as an additional accessory.
Analog Outputs
WARNING: The analog outputs are not protected against any input voltage! Any voltage
applied to the analog outputs can cause irreversible damage of the circuit.
RS232 Interface
The unit uses special interface cable. Another cable can cause the units malfunction.
Optical Output (ST)
The ST connector is a high precision optical component. Please keep it clean and dry.
Always use the rubber cap to close the output when not in use.

9.4

Operation Notes

Measurement
To achieve the highest accuracy pH-1 micro should be warmed-up for 5min before starting
the measurement. Please see the details of measurement process described in pH-1 micro
manual.
Temperature Compensation
No other than supplied temperature sensor could be used with the unit. The use of any other
temperature sensor can damage the device.

Concluding Remarks

47

10 Concluding Remarks
Dear customer,
With this manual, we hope to provide you with an introduction to work with the pH-1 micro
fiber-optic phase detection device.
This manual does not claim to be complete. We are endeavored to improve and supplement
this Version.
We are looking forward to your critical review and to any suggestions you may have.
You can find the newest version at www.loligosystems.com
With best regards,

Your Loligo Team

48

Appendix

11 Appendix
11.1 Basics in Optical Sensing
11.1.1

Major Components of Fiber-Optic Microsensors

In optical chemical sensors, the analyte interacts with an indicator and changes its optical
properties. The result is either a change in the color (absorbance or spectral distribution) or
the luminescence properties (intensity, lifetime, polarisation). Light acts as the carrier of the
information.
The major components of a typical fiber optical sensing system are

a light source to illuminate the sensor (laser, light emitting diode, lamps)
an optical fiber as signal transducer (plastic or glass fiber)
a photodetector (photodiode, photomultiplier tube, CCD-array)
the optical sensor (indicator immobilised in a solid matrix)
glass-fiber
with its sensor tip

OF

ST

syringe needle

LEDsig

silica fiber coupler

sensor housing

PMT

ST

microoptode

LEDref

Figure 11.1 Schematic drawing of the optical setup of

a measuring system with Microsensors
(LED: light emitting diodes, PMT: photomultiplier, OF:
optical filters, ST: fiber connector)

11.1.2

Figure 11.2 Scheme of a sensor

Luminescence Decay Time

The pH-1 micro measures the luminescence decay time of the immobilised luminophore as
the analyte dependent parameter.
= f([O2])

(1)

The pH-1 micro uses the phase-modulation technique to evaluate the luminescence decay
time of the indicators. If the luminophore is exited with a sinusoidally intensity modulated
light, its decay time causes a time delay in the emitted light signal. In technical terms, this
delay is the phase angle between the exiting and emitted signal. This phase angle is shifted
as a function of the analyte concentration. The relation between decay time and the phase
angle is shown by the following equation:

tan
2 f mod

(2a)

tan = 2 f mod

(2b)

tan f([O2])

(2c)

: luminescence decay time; : phase angle; fmod: modulation frequency

49

Appendix
I/Imax

I/Imax

1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

reference signal

1
0
1

0
-5

10 15
time [s]

20

25

Figure 11.3 Schematic of the single

exponential decay (t0 > t1).

11.1.3

30

-5

measuring
signal

10 15 20
time [s]

25

30

Figure 11.4 The luminophore is excited with sinusoidally

modulated light. Emission is delayed in phase expressed by the
phase angle F relative to the excitation signal, caused by the
decay time of the excited state

Advantages of Optical Lifetime Based Sensors

the signal is independent of changes in flow velocity;

they are insensible towards electrical interferences and magnetic fields;
long-term stability and low drift;
using silica fibers, it is possible to measure in samples while physically separate from the
light source and detectors;
light conducting fibers are able to transport more information than power currents
(information can be simultaneously transferred, e.g., intensity of light, spectral distribution,
polarisation, information such as decay time or delayed fluorescence);

The measurement of the luminescence decay time, an intrinsically referenced parameter,

has the following advantages compared to the conventional intensity measurement.
The decay time does not depend on fluctuations in the intensity of the light source and the
sensitivity of the detector;
The decay time is not influenced by signal loss caused by fiber bending or by intensity
changes caused by changes in the geometry of the sensor;
The decay time is, to a great extent, independent of the concentration of the indicator in
the sensitive layer Photobleaching and leaching of the indicator dye has no influence
on the measuring signal;
The decay time is not influenced by variations in the optical properties of the sample
including turbidity, refractive index and coloration.

50

Appendix

The measurement of the luminescence decay time, an intrinsically referenced parameter,

has the following advantages compared to the conventional intensity measurement.
The decay time does not depend on fluctuations in the intensity of the light source and the
sensitivity of the detector;
The decay time is not influenced by signal loss caused by fiber bending or by intensity
changes caused by changes in the geometry of the sensor;
The decay time is, to a great extent, independent of the concentration of the indicator in
the sensitive layer Photobleaching and leaching of the indicator dye has less influence
on the measuring signal;
The decay time is not influenced by variations in the optical properties of the sample
including turbidity, refractive index and coloration.
11.1.4

Dynamic Quenching of Luminescence

The principle of measurement is based on the effect of dynamic luminescence quenching by

molecular oxygen. The following scheme explains the principle of dynamic luminescence
quenching by oxygen.

emission of
light

1
absorption of light

excited
state

energy transfer
by collision
no emission of light

Figure 11.5 Principle of dynamic quenching of luminescence by molecular oxygen

(1) Luminescence process in absence of oxygen
(2) Deactivation of the luminescent indicator molecule by molecular oxygen

The collision between the luminophore in its excited state and the quencher (oxygen) results
in radiationless deactivation and is called collisional or dynamic quenching. After collision,
energy transfer takes place from the excited indicator molecule to oxygen which
consequently is transferred from its ground state (triplet state) to its excited singlet state. As
a result, the indicator molecule does not emit luminescence and the measurable
luminescence signal decreases.
A relation exists between the oxygen concentration in the sample and the luminescence
intensity as well as the luminescence lifetime which is described in the Stern-Volmerequation (1). Here, 0 and are the luminescence decay times in absence and presence of
oxygen (I0 and I are the respective luminescence intensities), [O2] the oxygen concentration
and KSV the overall quenching constant

51

Appendix

I0 0
=
= 1 + K SV [O 2 ]

I
I = f([O 2 ])

(3)

= f([O 2 ])
I:
I0:
:
0:
KSV:

Luminescence intensity in presence of oxygen

Luminescence intensity in absence of oxygen
Luminescence decay time in presence of oxygen
Luminescence decay time in absence of oxygen
Stern-Volmer constant (quantifies the quenching efficiency and therefore the sensitivity
of the sensor)
[O2]: oxygen content
1.0

(B)

(A)

0.8

0.6

I0/I or 0/

I/I0 or /0

0.4
2
0.2

20

40

60

80

1
100

oxygen content [%]

Figure. 11.6 (A) Luminescence decrease in the presence of oxygen. (B) Stern-Volmer plot.

Indicator dyes quenched by oxygen are, for example polycyclic aromatic hydrocarbons,
transition metal complexes of Ru(II), Os(II) and Rh(II), and phosphorescent porphyrins
containing Pt(II) or Pd(II) as the central atom.
11.1.5

Dual Lifetime Referenced (DLR) Optical Sensors

The measurement of intensity is simple in terms of instrumentation but its accuracy is often
compromised by adverse effects such as drifts of the opto-electronic system and variations
in the optical properties of the sample including fluorophore concentration, turbidity,
coloration and refractive index. Therefore, efficient referencing methods are required for
quantification of intensity signals. Among those, ratiometry, i.e., the measurement of the
fluorescence intensity at two or more wavelengths of a single indicator fluorophore or an
indicator fluorophore plus an inert fluorescence standard, is common to reference
fluorescence intensity. However, this method requires two separate optical channels thus
complicating the optical setup. For example, the drift in the sensitivity of both channels can

52

Appendix

be different, as can be the intensities at two excitation wavelengths. Light scatter and signal
loss caused by fiber bending (e.g. in fiber optic sensors or certain sensortiterplate readers)
further contribute to effects not compensated by two-wavelength referencing.
Alternatively, the measurement of the fluorescence decay time, an intrinsically
referenced parameter, is hardly affected by fluctuations of the overall fluorescence intensity.
The decay time of most pH-sensitive indicator dyes, however, is in the nanosecond time
scale requiring a sophisticated and expensive instrumentation which limits the use in sensor
application.
We use new and general logic to reference fluorescence intensity signals by decay
time measurement. In contrast to the most common ratiometric method, where
luminescence excitation or emission is measured at two wavelengths, this scheme uses a
couple of luminophores with different decay times and similar excitation spectra. An analyteinsensitive s-lifetime luminophore is combined with an analyte-sensitive ns-lifetime
fluorophore, and a method is presented how to convert fluorescence intensity into a phase
shift. Preferably, the reference dyes display decay times in the sensorsecond or millisecond
time domain to simplify the opto-electronic system.
The phase-modulation (frequency-domain) method is a well-established technique for the
measurement of luminescence decay times and was described in chapter 12.1.4. The phase
angle measured at a single modulation frequency (fmod) reflects the luminescence decay
time () provided that the decay is single-exponential (equation 4):

tan
2f mod

(4)

In this scheme, two luminophores are used. The first, referred to as the indicator, has a short
decay time (ind), the second acting as the reference standard has a decay time in the s
range (ref). Ideally, the two luminophores have overlapping excitation and emission spectra
so that they can be excited at the same wavelength and their fluorescence can be detected
using the same emission window and photodetector. The phase shift m of the overall
luminescence obtained at a single frequency depends on the ratio of intensities of the
reference luminophore and the indicator dye. m can be represented as the superposition of
the single sine wave signals of the indicator and the reference luminophore (see Figure
11.7).
The reference luminophore gives a constant background signal (ref) while the
fluorescence signal of the indicator (ind) depends on the analyte concentration. The average
phase shift m directly reflects the intensity of the indicator dye and, consequently, the
analyte concentration. The modulation frequency is adjusted to the decay time of the
reference dye.
2

overall signal
reference

(A)

amplitude

amplitude

indicator
(=LED frequency)

-1

(B)

overall signal
reference

1
0
indicator (=LED)

-1
ind m
ref

ind m ref

-2

-2
0

45

90

135

180

m []

225

270

315

360

45

90

135

180

m []

225

270

315

360

Figure 11.7 Phase shift of the overall luminescence (m), the reference (ref) and the indicator (ind).
Fluorescence of the indicator in (A) absence and (B) presence of the analyte.

Equations 5 and 6 show the superposition of the phase signals of the reference dye,
which possess a constant decay time and luminescence intensity, and the indicator:

53

Appendix

A m cos m = A ref cos ref + A ind cos ind

A m sin m = A ref sin ref + A ind sin ind

(5)

(6)
where A is the amplitude of either overall signal (m), luminophore (ref), or indicator (ind),
and is the phase angle of either the overall signal (m), the luminophore (ref), or the
indicator (ind), respectively. In case the modulation frequency (fmod) is optimal, tan ref is
described by equation 7
tan ref = 2 f mod ref = 1
(7)
and ind can be written as

tan ind = 2 f mod ind =

2 ind ind
=
2 ref ref

(8)

The reference luminophore has a decay time that is orders of magnitude longer than that of
the indicator. Consequently, ind can be set equal to zero in equation 9, since at low
modulation frequencies (in the kHz range) there is no phase shift.

tan ind =

ind ind << ref

0 ind 0
ref

(9)

The decay time of the reference luminophore is not affected by the analyte, hence:
ref = constant tan ref = constant ref = constant

(10)

Therefore, equations 5 and 6 can be simplified to give 11 and 12, respectively:

A m cos m = A ref cos ref + A ind

A m sin m = A ref sin ref

(11)
(12)

Dividing equation 11 by 12 results in a correlation of the phase angle (m) and the intensity
ratio of the indicator dye (Aind) and reference luminophore (Aref) :

A cos ref + A ind

A
A m cos m
1
= cot m = ref
= cot ref +
ind
A m sin m
A ref sin ref
sin ref A ref

(13)

In this equation, cotm reflects the referenced intensity of the fluorescence indicator. A linear
relation is obtained between cot (m) and the ratio of Aind/Aref, because the phase angle of
ref of the reference luminophore was assumed to be constant. This method is referred to as
Dual Lifetime Referencing (DLR).
11.1.6

Fluorescence (Frster) Energy Transfer (FET)

The term fluorescence energy transfer refers to a non-radiative transfer of excited state
energy from a donor (D) to an acceptor (A) and results from a dipole-dipole interaction
between donor and acceptor. Non-radiative energy transfer does not involve the emission
and reabsorption of photons. A transfer, where the acceptor dye reabsorbs photons emitted
by the donor is called radiative transfer or inner filter effect. The rate of non-radiative energy
transfer (kT) depends on the fluorescence quantum yield of the donor, the overlap of the
emission spectrum of the donor with the absorption spectrum of the acceptor, and their
relative orientation and distance. The theory was derived by Frster, who gave a quantitative
expression of kT between a donor and acceptor pair at a fixed separation
distance r (equation 14).

54

Appendix

8.71 10 23 2 d
1 R
J = 0
kT =
6
4
d r
r n d

(14)

with

J = FD () A () 4 d

(15)

where d and d are the quantum yield and lifetime of the donor in absence of the acceptor,
n is the refractive index of the medium, r is the distance between donor and acceptor and 2
is a factor describing the relative orientation in space of the transition dipoles of the donor
and acceptor. The overlap integral J, represented in equation 15, expresses the degree of
spectral overlap between donor emission and the acceptor absorption. Fd() is the corrected
fluorescence intensity of the donor in the wavelength range to +d with the total intensity
normalized to unity and A() is the extinction coefficient of the acceptor at . The Frster
distance R0 is the donor-acceptor critical transfer distance at which radiative decay and nonradiative energy transfer are equally probable. Remarkably, the efficiency depends on the 6th
power of r.
Many optical sensors exploit the principle of energy transfer. The potential of
ET-based sensors relies on the fact that well-investigated absorbance based indicators can
be applied by adding an analyte-insensitive fluorescent donor with a sufficiently large
spectral overlap. Sensors for pH, carbon dioxide and ammonia have been realized by energy
transfer from a pH-insensitive donor to a pH-sensitive acceptor. Additionally, luminescence
energy transfer is a convenient way to overcome the lack of suitable lifetime based
indicators since the color change of an absorber can be converted into a decay time
information.
Energy transfer measurements require a constant separation distance between the
D-A pair which does not vary during the excited state lifetime of the donor. This can be
achieved by covalently linking donor and acceptor via spacer groups. An alternative way to
fix the separation distance is the formation of donor-acceptor ion-pairs.

11.1.7

Literature

If you want to find out more about this subject, we recommend the following publications.
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Appendix

55

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